IL-4 inhibits the expression of high affinity IL-2 receptors on monoclonal human B cells.

In the presence of anti-mu antibodies (anti-microAb), monoclonal B lymphocytes from patients suffering from B type chronic lymphocytic leukemia (B-CLL) can respond to IL-2. In contrast to the effect it exerts on normal B cells, IL-4 does not promote DNA synthesis by B-CLL lymphocytes. Rather this interleukin inhibits the response to IL-2 in all patients' cells that responded to this interleukin. We thus examined whether IL-4 would modulate the number and/or the affinity of IL-2 receptors. A 3-day activation of cells by anti-microAb induced a few hundred high affinity IL-2 receptors (HA-IL-2R) on B-CLL cell surface, as determined by Scatchard analysis. Treatment of cells with IL-4 caused a marked decrease in the number of HA-IL-2R without interfering with the binding ability of IL-2. In contrast with this profound suppressive effect, IL-4 did not down-regulate the expression of each chain, alpha and beta (p55 and p75, respectively), of the HA-IL-2R heterodimer. In fact, the expression of alpha and beta induced by anti-microAb was enhanced by IL-4. Altogether, IL-4 exerts a critical influence on the function and the configuration of HA-IL-2R without inhibiting the expression of two subunits, alpha and beta.

Immunohistological studies of surface antigen on colonic lymphoid cells in normal and inflamed mucosa. Comparison of follicular and lamina propria lymphocytes.

Mucosal samples from 16 patients with idiopathic inflammatory bowel disease were examined immunohistologically using several monoclonal antibody combinations, and the results were compared with those obtained in other colonic inflammatory disorders and in normal mucosa. Within and around lymphoid follicles, most T cells expressed the restricted common leucocyte antigen (CD45R, displayed by unprimed T cells). Conversely, most lamina propria T cells were negative for CD45R but stained positively with UCHL1 (a monoclonal antibody recognizing an antigen displayed by primed T lymphocytes). The proportions of T-lymphocyte subpopulations in normal and inflamed mucosa were similar, except that CD6-negative, CD7-positive cells were significantly more frequent in inflammatory bowel disease. A characteristic feature of ulcerative colitis and Crohn's colitis was marked infiltration by CD45R+ lymphoid cells that did not coexpress T- or B-cell surface antigens; some stained positively with plasma cell reagents, suggesting that they may be activated B cells. The observations in the colitis sections are consistent with the interpretation that T and B cells alter their surface antigen expression as they emerge from follicles and enter the inflamed lamina propria.

Evaluation of CD5 and other differentiation antigens on human immunoglobulin-secreting cells using a combination of immunobead rosetting and reverse haemolytic plaque formation.

This paper describes a new method, with high specificity and sensitivity, for evaluating cell surface markes such as differentiation antigens and cytokine receptors on immunoglobulin-secreting cells. Mononuclear cells, freshly derived from peripheral blood or following stimulation in vitro with pokeweed mitogen or Staphylococcus aureus Cowan I, are partially depleted of T cells and monocytes using immunomagnetic beads (Dynabeads) coated with anti-CD2. The cells are incubated with Dynabeads coated with monoclonal antibody against the cell marker under investigation and then used in a protein A haemolytic plaque assay. Plaque-forming cells (PFC) with (marker-positive) and without (marker-negative) attached beads can be readily enumerated. Values are given for percentages of IgG-, IgA- and IgM-PFC bearing CD19, CD38, CD25 and CD5.

Association of Ig L chain-like protein lambda 5 with a 16-kilodalton protein in mouse pre-B cell lines is not dependent on the presence of Ig H chain protein.

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.

Immune activation and autoantibodies in humans with long-term inhalation exposure to formaldehyde.

Four groups of patients with long-term inhalation exposure to formaldehyde (HCHO) were compared with controls who had short-term periodic exposure to HCHO. The following were determined for all groups: total white cell, lymphocyte, and T cell counts; T helper/suppressor ratios; total Ta1+, IL2+, and B cell counts; antibodies to formaldehyde-human serum albumin (HCHO-HSA) conjugate and autoantibodies. When compared with the controls, the patients had significantly higher antibody titers to HCHO-HSA. In addition, significant increases in Ta1+, IL2+, and B cells and autoantibodies were observed. Immune activation, autoantibodies, and anti-HCHO-HSA antibodies are associated with long-term formaldehyde inhalation.

Nonneoplastic and nonhyperplastic thymus in myasthenia gravis. An immunohistochemical study with double immunoenzymatic labeling of basement membrane and cellular components.

Histologically normal thymus (type A) in patients with myasthenia gravis (MG) was immunohistochemically compared with hyperplastic MG thymus (type B) and normal non-MG thymus. In formalin-fixed, paraffin-embedded sections of ten type A, ten type B, and eight non-MG cases, the thymic epithelium and other cellular components were stained in conjunction with the basement membrane by a double immunoenzymatic method. This technique demonstrated a moderate architectural disturbance in type A thymus, with distended perivascular space (PVS), elongated medullary epithelium, and disrupted basement membrane. These changes were more prominent in type B thymus but were minimal to lacking in non-MG thymus. Compared with those in non-MG thymus, the myoid cells in MG thymuses of both types tended to cluster around the Hassall's corpuscles, with a slight decrease in number in type B but not in type A. B-lymphocytes were present in type B, type A, and non-MG thymuses in that order of abundance; the cells were confined to the medullary parenchyma in the non-MG group but were numerous both in the PVS and medulla in the MG groups. T-lymphocytes were increased in the expanded PVS of type A and B MG thymuses. The number of interdigitating reticulum cells was similar in the three groups, but the cellular distribution was more dispersed in MG thymuses of both types. These findings, although previously described in type B thymus, have not been well recognized in type A thymus. They support the view that a common abnormality (presumably chronic thymitis), differing in degree only, underlies MG thymuses regardless of the presence of follicular hyperplasia.

Lamins A and C are not expressed at early stages of human lymphocyte differentiation.

Lamins are major proteins of the nuclear envelope that are members of the intermediate filament protein family. In vertebrates, nuclei from differentiated tissues usually contain both lamins of the A and B subtypes, while embryonic tissues contain the B-type lamin only. We have examined the composition of the nuclear lamina in human B and T lymphocytes representative of distinct stages of lymphoid differentiation. We show here that, in both cell lineages, while lamin B is constitutively expressed at all stages of differentiation, A-type lamin expression is restricted to later developmental stages.

Rapid method for distinguishing clonal from polyclonal B cell populations in surgical biopsy specimens.

The polymerase chain reaction (PCR) was used to detect clonal rearrangements of the immunological heavy chain gene in frozen samples of human lymphoid tissue. DNA sequences in rearranged genes were amplified using oligomeric primers predicted from conserved sequences in the variable (VH) and joining (JH) regions. On polyacrylamide gel electrophoresis, polyclonal B cell proliferations showed a "smear", probably due to the variable lengths of the diversity (DH) region genes and the N regions separating the VH and DH and JH regions. In contrast, DNA from B cell lymphomas showed a clear single band in eight out of 10 cases. PCR undertaken on germ line DNA from non-lymphoid tumours showed no detectable bands or smears. The method can be completed within one day of biopsy, compared with several days in the case of conventional DNA blot analysis. Furthermore, it is cheaper, simpler, avoids the need for radioactive materials and requires very small amounts of DNA (about 1 micrograms).

[Presence of a non-HLA antigen in B-lymphocytes from patients with rheumatic fever and their relatives defined using monoclonal antibodies]. / Presencia de un antígeno no-HLA en los linfocitos B de pacientes con fiebre reumática y sus familiares definido mediante anticuerpos monoclonales.

Numerous investigators have suspected that there is a genetic predisposition to rheumatic fever (RF). In this context a group of investigators at Rockefeller University have produced a monoclonal antibody that identifies an antigen present in 100% of all RF studied at that center. Using this antibody, labeled D8/17, we studied 47 patients with acute rheumatic fever and rheumatic heart disease. Of these, 39 were not receiving steroids at the of the test and 35 were positive for the marker (89.7%). The highest percentage of positive cells was seen in the probands with 34.6 +/- 13.16%, while unaffected mothers, fathers and siblings gave 24.9, 5.2 and 7.3% respectively. The control group had an average of 7.5% of positive cells. This study and previous ones, performed by the Rockefeller University group in which HLA typing was included, suggest an autosomal recessive mode of inheritance, not associated with the MHC system, for the D8/17 antigen. Rheumatic fever; non-HLA antigen in; genetic predisposition in.

Characterization of peripheral blood and salivary gland lymphocytes in Sjögren's syndrome.

Primary Sjögren's syndrome (SS) is an autoimmune disease resulting from lymphocyte infiltration of lacrimal and salivary glands (SG). This study was designed to investigate the peripheral blood (PBL) and SG lymphocytes in 14 patients with primary SS and control subjects. With the use of monoclonal antibodies, cells were stained to identify T-cells and T-cell subsets (T-helper and T-suppressor) and cells positive for HLA-DR antigen, whereas B cells were determined by the Smlg (surface membrane immunoglobulin) method. Lymphocytes in SG biopsy specimens were characterized by means of monoclonal antibodies and the immunoperoxidase technique. In the peripheral blood lymphocytes, there was a significant reduction in T cells and suppressor T cells. T lymphocytes and mostly helper T cells were predominant around the ducts and within the lymphocytic infiltrates in the minor SG biopsy samples of patients with SS. Suppressor T cells and B cells were found in fewer numbers, HLA-DR(+) cell populations had increased, and IgG- and IgA-bearing plasma cells were also present within the infiltrates. These results may contribute to our understanding of the immunopathogenesis of primary SS.

Cell-type-specific control elements of the lymphotropic papovavirus enhancer.

Lymphotropic papovavirus (LPV) exhibits a highly restricted host range, in which only cells of primate B-lymphocyte origin are permissive for infection. Its enhancer element contributes to this tropism, since transcriptional potentiation is confined to cells of the hematopoietic lineage. Nuclear extracts from B and T cells, but not from HeLa cells, contain protein factors that interact specifically with the LPV 63-base-pair enhancer repeat, as demonstrated by DNase I footprinting and gel retardation experiments. Within the repeat three sequence motifs were identified: the core motif, the Pu box, and a novel element named T motif. Functional analysis demonstrated that these motifs as well as some sequences upstream of the repeat contribute to the optimal activity of the enhancer. There are clear differences between the patterns of binding of the B and T lymphocyte nuclear proteins to the enhancer which are also reflected in the transcriptional activity of the enhancer in both cell types. Furthermore, the activity of the LPV enhancer and its interaction with nuclear proteins seem to be regulated during B-cell differentiation.

The distribution of T and B lymphocyte populations and MHC class II expression in human fetal and postnatal intestine.

The development of the intestinal mucosal immune barrier is an important protective adaptation for postnatal life. The distribution and phenotype of T and B lymphocytes in human fetal intestine and lymphoid tissues have been characterized and compared to the distribution and phenotype of lymphocytes in postnatal intestine. The characterization of lymphocyte phenotype and MHC class II antigen distribution was done using MAb and an avidin-biotin complex immunohistochemical staining technique. Intraepithelial lymphocytes were occasionally present in fetal intestine and were primarily CD3+, CD8+. T lymphocytes were readily identified in the lamina propria of fetal intestine, but most were clustered in lymphoid aggregates. Cells identified by anti-IgA1 and anti-IgA2 were the most numerous cells of B cell lineage in the lamina propria of postnatal intestine, whereas IgM+ and IgD+ lymphocytes predominated in fetal tissues. However, IgA-bearing cells were identified in lymphoid aggregates of the intestine or spleen of some fetuses. This finding suggests that B lymphocytes can undergo Ig switching in utero. Additionally, fetal intestinal epithelial cells did not express MHC class II antigens, unlike some postnatal intestinal tissues. It is possible that postnatal events such as antigen exposure may be important for the induction of these class II antigens on intestinal epithelial cells.

Monoclonal antibody HML-1, a marker for intraepithelial T cells and lymphomas derived thereof, also recognizes hairy cell leukemia and some B-cell lymphomas.

Current views regard monoclonal antibody HML-1 as an exquisite marker for intraepithelial T cells and primary intestinal and cutaneous T-cell lymphomas. We show that HML-1 reacted with 11 of 12 cases of hairy cell leukemia, with 1 of 13 cases of primary gastrointestinal B-cell lymphoma, and with an unclassified large-cell B lymphoma of the thoracic wall. We conclude that HML-1 is not restricted to the T-cell lineage and that the HML-1 antigen is expressed in a small subset of both T- and B-cell neoplasms.

A biologic assay for IL-4. Rapid fluorescence assay for IL-4 detection in supernatants and serum.

Interleukin-4 (IL-4) is a lymphokine produced by activated T helper cells with effects on T cells, B cells, monocytes and mast cells. The conventional tonsillar B cell assay used for quantification of IL-4 activity is sensitive to the presence of other cytokines and requires the acquisition of fresh cells on a regular basis. We have evaluated the ability of IL-4 in the presence of antibody to IgM to induce CD23 on a cultured B cell line (Ramos) The requirements of measuring hundreds of samples at a single time precluded ready use of a conventional flow cytometer. We therefore developed a rapid fluorescence assay which allows the detection of IL-4 in supernatants and serum. The use of a particle fluorescence immunoassay reproducibly allows detection of IL-4 to 6 U in supernatants and serum. This assay is specific for IL-4 and is not sensitive to other recombinant cytokines including IL-1, IL-2, IL-3, IL-6; interferon-alpha, -beta or -gamma, tumor necrosis factor (TNF) or GM-CSF. Finally, in cancer patients receiving IL-4 its detection in serum using this assay reveals an alpha distribution phase of approximately 8 min and a beta clearance phase of approximately 48 min.

Peripheral B-lymphocyte markers and function in IgA nephropathy.

Peripheral blood B-lymphocyte markers and functions were observed in 21 patients with IgA nephropathy (IgA NP) and in 16 controls. IgA NP B lymphocytes expressed significantly higher positivity with Leu 1 (CD 5) monoclonal antibody than controls. CD 5 positive B lymphocytes are thought to be a distinct subset of the B cells (autoregulatory B lymphocytes) inducible in IgA NP by lipopolysaccharide (LPS) stimulation parallel to the higher expression of surface IgM heavy chain positivity. The activated state of IgA NP B lymphocytes has been proved by their higher OKIa (HLA-DR) positivities but lower IOB1a (CD 21, C3d-receptor) and decreased IgG-Fc-receptor (ox-rosette) expression. IgA NP B lymphocytes showed a higher IgA but also IgG and IgM polyclonal immunoglobulin production than control B lymphocytes in co-cultures with T lymphocytes. Not only regulatory T lymphocyte subsets but also serum derived from IgA NP patients stimulated the immunoglobulin production of IgA NP B cells.

Millimolar concentrations of ascorbic acid in purified human mononuclear leukocytes. Depletion and reaccumulation.

Ascorbic acid (vitamin C) was found in isolated human mononuclear leukocytes and their purified components in millimolar concentration. Intracellular ascorbic acid was depleted greater than 96% during cell culture and was rapidly reaccumulated after addition of physiologic concentrations of ascorbic acid to the extracellular medium. Purified cells maintained concentration gradients of ascorbic acid as large as 100-fold across the plasma membrane. The ability to vary intracellular ascorbic acid concentrations over such a wide range makes it possible for the first time in these cells to study ascorbic acid function in direct relationship to intracellular concentration.

A transient acidification linked to intracellular Ca2+ in anti-mu-stimulated human B-lymphocytes.

Mitogen-induced cellular proliferation is in many cell types preceded by rapid changes in intracellular pH and free Ca2+ concentration. We studied the patterns of pH and Ca2+ changes in normal resting human B-lymphocytes after exposure to anti-mu antibodies and the monoclonal antibody IF5, reactive with the CD20 antigen, both able to activate resting B-lymphocytes to enter the G1 phase of the cell cycle. Monitoring intracellular pH with the pH-sensitive, fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein, we demonstrated that poly- and monoclonal anti-mu antibodies induced a rapid (maximum change within 2 min) intracellular acidification of 0.06 pH units followed by a slower (10-15 min) alkalinization towards, or slightly above, the resting pH of 6.88. The acidification response was amiloride-resistant, whereas the return to baseline was sensitive. Intracellular free Ca2+ was measured by using the fluorescent Ca2+ dye, indo-I. Exposure of cells to anti-mu resulted in a rapid increase (maximum change within 2 min) in cytoplasmic Ca2+ of 340 nM and a slower decline in fluorescence back to baseline of about 180 nM. In contrast to anti-mu, IF5 caused no change in cytoplasmic Ca2+ and pH. However, the Ca2+ ionophore ionomycin at low concentrations mimicked the Ca2+ response as well as the pH response to anti-mu. In Ca2(+)-free solutions the intracellular Ca2+ stores are usually rapidly depleted and, indeed, the Ca2+ and pH responses to anti-mu were reduced after 5 min and almost abolished after 35 min under such conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Inmunidad y Candida albicans: un estudio de poblaciones y subpoblaciones linfocitarias / Immunity and Candida albicans: a population and linfocitary sub-population study

En este trabajo se presentan los resultados obtenidos en un estudio inmunológico, realizado en un grupo de 13 pacientes con síndrome de hipersensibilidad a la Candida. Los estudios micológicos confirmaron los hallazgos clínicos de candidiasis. En cada uno de los casos se realizó cultivo, tipificación y recuento de colonias, que oscilaron entre 9.800 y 90.000 unidades formadoras de colonias de C. albicans/g de materia fecal peso húmedo. Las pruebas de hipersensibilidad cutánea fueron realizadas con 4 antígenos. Todos los pacientes mostraron positividad en la lectura precoz y negatividad en la tardía a excepción de 4 de ellos. La inmunoglobulina E fue cuantificada por ELISA y se encontraron valores por encima de lo normal en el 84% de los casos. Los valores hallados oscilaron entre 31 y 1000 Ul/ml. El número de LB fue normal en todos los casos mientras que el de LT fue normal en el 46% y disminuídos en el 54% de los pacientes. En ambas determinaciones se usó el método de las rosetas. Las subpoblaciones de LT se determinaron por técnica de inmunofluorescencia indirecta, con anticuerpos monoclonales. Los CD4+ (cooperadores/inductores) se encontraron disminuídos en el 77% de los casos mientras que los CD8+ (citotóxicos/supersores) estuvieron normales en la mayoría de ellos (84%). De acuerdo a estos resultados se puede deducir que existe una estrecha relación entre candidiasis, IgE aumentada e inmunodeficiencia fundamentalmente de la inmunidad mediada por células y en especial de las ...

Determinación de poblaciones y subpoblaciones linfocitarias en pacientes con enfermedad respiratoria inmunoalergica

Se determinaron los niveles de IgE séricos y se cuantificaron las poblaciones de linfocitos B (LB) y (LT) y las subpoblaciones T4 (CD4+) y T8 (CD8+) en 20 pacientes con enfermedad respiratoria inmunoalérgica (rinosinusitis y/o asma bronquial). Los niveles de IgE se encontraron aumentados en el 90% de los pacientes (valor promedio 412 UI/ml; rango 56-1000 Ul/ml). El recuento de LB estuvo normal o disminuído y en cuanto a la población de LT, se observó la existencia de una relación entre niveles bajos de IgE y LT normales y niveles altos de IgE con LT bajos. La subpoblación de linfocitos T8 estuvo normal en el 50% de los pacientes estudiados. Fueron significativos los resultados obtenidos al estudiar la subpoblación T4, que se encontró disminuída en el 80% de los pacientes. Quizás un estudio discriminativo de las subclasses que la constituyen, explicaría los hallazgos inmunológicos en estos pacientes. De acuerdo a estos resultados se puede concluir que es muy importante el estudio del sistema inmune del paciente antes de implementar la inmunoterapia específica, tratando de emplear un agente terapéutico que normalice la población de linfocitos afectada y conseguir el mayor éxito en el tratamiento

Prognostic significance of flow cytometry studies in B-cell non-Hodgkin lymphoma.

The prognostic relevance of flow cytometric DNA measurements of lymph node biopsies in relation to histopathology according to the Kiel classification, stage, age and presence or absence of B-symptoms was investigated in 106 patients with non-Hodgkin lymphomas (NHL) of proven B-cell type. Biopsies were taken at diagnosis before treatment. The mean proportion of cells in S-phase was significantly lower in the 76 patients with a 'low grade' NHL (4.4 per cent) than in the 30 patients with a 'high grade' NHL (13.0 per cent) (p less than 0.0001). High S-phase rates were associated with a poor prognosis within the whole material (p less than 0.01) and within the group of 'high grade' NHL (p less than 0.05). In the subgroup CB-CC lymphomas of 'low grade' NHL, the prognostic value of the S-phase rate was stronger than any other investigated parameter (p less than 0.05). In multivariate analyses, the S-phase rate gave, in several subgroups, independent prognostic information, besides clinico-pathological variables.